Archives of Environmental Health: An International Journal

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Erythrocyte Protoporphyrin Test for Occupational Lead Exposure Katsumaro Tomokuni MD , Ikuko Osaka MS & Masana Ogata MD To cite this article: Katsumaro Tomokuni MD , Ikuko Osaka MS & Masana Ogata MD (1975) Erythrocyte Protoporphyrin Test for Occupational Lead Exposure, Archives of Environmental Health: An International Journal, 30:12, 588-590, DOI: 10.1080/00039896.1975.10666785 To link to this article:

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Erythrocyte Protoporphyrin Test for Occupational Lead Exposure

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Katsumaro Tomokuni, MD; Ikuko Osaka, MS; Masana Ogata, MD • Determination of erythrocyte protoporphyrin levels was performed in workers who are occupationally exposed to lead. The level of erythrocyte protoporphyrin was easily measured with microsamples of blood by utilizing a fluorescent assay. The log of erythrocyte protoporphyrin level was closely correlated to blood lead level (r=.72) in leadexposed workers. The erythrocyte protoporphyrin test is especially useful in the detection of mild increases in blood lead concentration under conditions of occupational exposure. (Arch Environ Health 30:588-590, 1975)


ead is known to inhibit several en-

.1....J zymes essential in heme biosynthesis. Of these enzymes, it has been considered that the inhibition of o-aminolevulinic acid dehydratase (ALAD) catalyzing the step from 0aminolevulinic acid (ALA) to porphobilinogen (PBG) and that of heme synthetase catalyzing the step from protoporphyrin IX to heme are of particular interest. l ' 3 For instance, erythrocyte ALAD activity is partially inhibited by lead, long before any other effects on the hematopoietic system are measurable,1'~ and the degree of inhibition closely correlates with blood lead levels. 7 ' l6 The ALAD test is, therefore, considered to be most reliable for use as a control in workers who are occupationally exposed to low concentrations of lead. In addition, it has been demonstrated that in occupational exposure to lead, erythrocyte ALAD activity undergoes much greater reductions, not only in the manifest phase of lead poisoning, but even at the very beginning of exposure 15 and ALAD activity remains reduced long after exposure has ceased. l7 ,l8 These investigations Submitted for publication Sept 23, 1974; accepted April 2, 1975. From the Department of Public Health, Okayama University Medical School, Okayama, Japan. Reprint requests to Department of Public Health, Okayama University Medical School, 25-1 Shikata Okayama, Japan (Dr Tomokuni).


Arch Environ Health-Vol 30, Dec 1975

may lead to the conclusion that the measuremen t of erythrocyte ALAD activity is not of primary importance in the medical surveillance of workers exposed to lead for a long period of time, though itt can be very useful as a rapid preliminary test. On the other hand, because of the enzyme inhibition described above, the measurement of erythrocyte protoporphyrin (EP) can also be expected to be more useful for the evaluation of the degree of lead effects in occupational exposure. Recently, Piomelli l9 developed a simple and rapid micromethod for the determination of EP levels. He has stated that EP concentration increases exponentially when blood lead concentration is increased. Sassa and associates 20 have also indicated that the EP test is especially useful in the detection of chronic subclinical lead poisoning in children. We performed the EP test to measure lead absorption in workers occupationally exposed to lead. In the present study, the results of the EP test in the exposed workers are discussed in relation to blood lead level, erythrocyte ALAD activity, and duration of exposure. SUBJECTS AND METHODS Sixty-five workers chosen as subjects were employed in a polyvinyl chloride factory where they were exposed to the dust of stabilizing agents consisting of a mixture of tribasic lead sulfate (approximately three parts of PbO and one part each of PbS0 4 and H 2 0) and lead stearate. The duration of occupational lead exposure ranged from 3 to 18 years. Surveys on alcohol ingestion were also performed among the workers. Approximately 40% of them use alcohol in the form of sake (Japanese rice beer), in quantities of 180 to 360 ml (mean-230 ml) per day. Sake contains approximately 15% pure alcohol, so the mean pure alcohol intake is about 35 ml per day, which places the workers in the classification of light drinkers. The determination of EP levels was performed fluorophotometrically according to

a slight modification of the Piomelli method. '9 Using a pipette, 0.05 ml of whole blood was placed into a lO-ml test tube containing 0.25 ml of 5% diatomaceous earth suspension in saline. We added 4 ml of an ethyl acetate-glacial acetic acid (4:1) mixture and agitated the tube using a vibration mixer for 10 seconds. After centrifuging at 3,000 rpm for five minutes, we poured the supernatant into a 15-ml glass (stoppered) tube. We added 4 ml of 1.5N HCI and shook the tube 80 times by hand to extract the EP. An aliquot of the lower HCI phase was transferred into another test tube with a capillary pipette and the fluorescence of the extract was measured using a corrected, recording spectrofluorophotometer. The excitation wavelength was set at 405 nm and the emission at 610 nm; both spectral band widths were maximallyopened (excitation, 19 nm; emission, 7.5 nm). A standard solution (0.05,ug/ml) was prepared from a preweighed vial containing 5,ug of ooproporphyrin 1. After adding 10 ml of 1.5N HCl to the vial, we heated it for three minutes in boiling water. Then, the 1:10 dilution was used as the standard with 1.5N Hel. The EP concentration was calculated in micrograms per 100 ml of erythrocytes (red blood cells [RBCsJ) from the following formula:




- - X - - - - X 0.05 X


4.8 - - X 100,


where Ht=hematocrit reading, Fu= fluorescence of unknown, Fs = fluorescence of standard (0.05f.Lg/ml), 1.11= conversion of fluorescence of coproporphyrin 1 in 1.5N HCI into fluorescence of protoporphyrin IX in the extract, 4.8 = final volume of HCl (increases from original, 4 ml). Erythrocyte ALAD activity was meaMeasurements Obtained From Workers Exposed to Lead

Blood lead, p.g/100 ml ALAD activity, p.mol PBG/hr/liter RBC Protoporphyrin, p.g/100 ml RBC Hemoglobin, gm/100 ml

Mean 36 340

SD 13 179

114 171 15.2 1.7

Erythrocyte Protoporphyrin Test-Tomokuni et al

anticoagulant. Iron deficiency anemia was not detected among the workers. Blood hemoglobin levels were determined by the standard method after conversion to cyanmethemoglobin. Hematocrit levels were measured with the use of a microcapillary centrifuge. Blood lead concentrations were determined using a flameless atomic

sured according to the Tomokuni procedure. 9 The enzyme assay was performed for one hour at pH 6.8. The enzyme activity was expressed as micromols of PBG per hour per liter of erythrocytes (RBes). Blood samples were collected in leadfree tubes containing a mixture of potassium oxalate and ammonium oxalate as an

absorption spectrophotometer equipped with a deuterium background corrector. RESULTS

Several biochemical measurements observed in the workers occupationally exposed to lead given in the Table. From the data on blood lead


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Erythrocyte protoporphyrin test for occupational lead exposure.

Determination of erythrocyte protoporphyrin levels was performed in workers who are occupationally exposed to lead. The level of erythrocyte protoporp...
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